3. Phase 0:
Rapid Depolarization
(Na+ influx)
Phase 1:
Early Repolarisation
(Inward Na+ current
deactivated,
Outflow of K+):
Transient Outward Current
Phase 2:
Plateau Phase
(Slow inward Ca2+ Current balanced by
outward delayed rectifier K+ Current)
Phase 3:
Late Repolarisation
(Ca 2+current inactivates,
K+ outflow)
Action Potential of Cardiac Muscle 3
4. Cardiac Arrhythmias
“Heart beats with an irregular or abnormal rhythm”
Cardiac depolarization that deviate from normal due to
abnormalities in,
site of origin of impulse: Disturbances in impulse
formation
Its rate or regularity or conduction: Disturbances in
impulse conduction
5. Disturbances in impulse formation
Cardiac Arrhythmias
Interval between
depolarization of
pacemaker cell
Duration
of Action
potential
Duration of
diastolic
interval
Affects the pacemaker rate
Early After depolarization
Transient depolarization that
interrupt phase 3
Eg. Long QT related arrhythmia
Delayed After depolarization
Transient depolarization that
interrupt phase 4
Intracellular calcium overload
Eg. Digitalis induced arrhythmia
6. Disturbances in impulse conduction
Cardiac Arrhythmias
Obstacle to
homogenous
conduction
Depressed Na or Ca current
7. Basic Pharmacology of anti-arrhythmic
agents
to reduce the ectopic pacemaker activity
modify the conduction/refractoriness in re-entry circuits
Sodium channel blockade
Blockade of sympathetic autonomic effects in heart
Prolongation of effective refractory period
Calcium channel blockade
8. Basic Pharmacology of anti-arrhythmic
agents
Use-dependent / State
dependent blockade
Drugs decrease automaticity of ectopic pacemakers more than
SA node
Reduce conduction and/or increase refractory period in
depolarized tissue than in normally polarized tissue
Drugs bind readily to activated channels (ie. during phase o) &
inactivated channels (phase 2) but poorly or not at all to resting
channels.
9. Vaughan & William’s classification
Class I Sodium channel blockers
Class IA: Prolong AP duration
Quinidine, Procainamide
Class IB: Shorten AP duration
Lignocaine, Mexiletine, Tocainide, Phenytoin
Class IC: No effect on AP duration
Encainide, Flecainide, Moricizine, Propafenone
Class II Beta Blockers (Esmolol, Metoprolol)
Class III Drugs prolonging ERP (Amiodarone,Sotalol )
Class IV Calcium channel blockers (Verapamil, Diltiazem)
10. Present indication & Need ?
Atrial fibrillation (AF) and atrial flutter
Acute setting: as an alternative to electrical cardioversion to
achieve SR
Chronic setting: to prevent recurrence
Ventricular arrhythmias:
As an adjunctive,
to improve the quality of life (QOL) of the patient
maximize battery life of defibrillator
Not successful in primary prevention of ventricular
arrhythmias in patients at risk of SD
Class I shown to increase mortality
Need for more effective and safe drugs
11. Evaluation methods
Pre-clinical Evaluation
Cell culture techniques: Studies on isolated
ventricular myocytes
In –vitro models
In- vivo models
Clinical Evaluation of anti-arrhythmic drugs
12. CELL CULTURE TECHNIQUE
Studies on Isolated Ventricular Myocytes
Oxygenated calcium free HEPES
buffered saline at 37º C at the rate 10
ml/min for 5 min
Same solution containing collagenase
and protease for 8 min
HEPES buffered saline containing 0.2
mM calcium chloride for 5 min
HEPES buffered
saline
Single cell
isolated
Ventricular
myocyte
13. Isolated myocyte stimulated electrically
Control period: frequency of 3Hz & AP recorded
Test drugs are added in the medium
Electrical stimulation after 10 min and
recording of AP
The change in AP amplitude before and after is compared
16. Anti-arrhythmic activity in isolated guinea
pig papillary muscle
The papillary muscle preparation transferred to organ bath and
experimental conditions maintained
Muscles are field stimulated to contract isometrically
( Voltage: twice threshold, frequency: 1 Hz and duration: 1 ms)
Equilibration of preparation for 90 min
Control measurements done during last 15 min of equilibration
Test drug added to the bath and post drug measurements taken after an
hour
Developed tension (DT), excitability (EX), and effective refractory
period (ERP) are measured.
17. Developed tension:
correlates with the Ca blocking activity of the drug.
Excitability:
measured by recording the strength of stimulus to generate a
response
correlates with the Na channel blocking action
Effective refractory period:
measured by continuous stimulation till a contraction is evoked
correlates with the K channel blocking action.
Anti-arrhythmic activity in isolated guinea
pig papillary muscle
18. Lagendorff Technique
Guinea pig anaesthetized
50 mg/kg sodium thiopentone
Mounted on the
dissection tray
The trachea cannulated and
secured with a thread to
ventilate the guinea pig
21. Lagendorff Technique
The incidence and duration of ventricular tachycardia or
ventricular fibrillation is recorded in the control as well as test
group
22. Acetylcholine or Potassium induced
arrhythmia
New Zealand White Rabbit (0.5 to 3 kg)
sacrificed and heart removed
After 5 min exposure to Ach/KCL, atria stimulated with rectangular
pulses (10 V, 0.75 ms duration, 400 – 1800 shocks/min)
Atria dissected and attached to an electrode
Control arrhythmia
6 – 10 min
3o min rest period Arrhythmia again induced
for 3 min
Test
drug
24. I
Lead I
between right and left foreleg
In axis of horizontal heart
ECG recording in animals
Lead II
between right foreleg & left hindleg
Along anatomical axis of heart
Lead III
between left foreleg & left hindleg
In axis of vertical heart
25. ECG recording in animals
Male SD rats weighing 250 -300 gram anaesthetizedRight jugular vein cannulated for injectionsECG recorded on polygraph at standard chart speed 100 mm/s
Variables measured:
Sigma T : Time for depolarization
wave to cross the atria
P –R interval
QRS interval
Q – T interval
RSh: height between peak of
R ans S wave
28. Aconitine antagonism in rats
Aconitine
Persistent activation of sodium channels resulting in arrhythmias
29. Aconitine antagonism in rats
Male Ivanovas rat weighing 300 – 400 g anaesthetized
Aconitine (5 mg/kg) dissolved in 0.1 N HNO3 administered
by continuous IV infusion at rate of 0.1 ml/min
Test drug given 5 min before start of Aconitine infusion orally
or intravenously
ECG in lead II recorded every 30 seconds
30. Aconitine antagonism in rats
Amount of Aconitine/100 g of
animal which induces,
Ventricular extrasystoles
Ventricular tachycardia
Ventricular fibrillation
Death
Higher dose of Aconitine in the treated group compared
to the untreated group: an indication of anti arrhythmic
activity
Procainamide 5 mg/kg
Lignocaine 5 mg/kg increased LD100
by 65%
31. Aconitine antagonism in rats
Scherf at al Atrial arrhythmia provoked by application of Aconitine
crystals to surface of right atrium in anesthetized dogs
McLeod &
Reynold Induced atrial arrhythmia in isolated rabbit atrium
Dadkar &
Bhattacharya
Used Aconitine as arrhythmogenic in conscious mice
Winslow Used Aconitine as arrhythmogenic in cats
32. Digoxin induced ventricular arrhythmia
Overdose of cardiac glycosides
induces,
ventricular extra systoles,
ventricular tachycardia,
ventricular fibrillation and death.
Occurrence of these symptoms can be delayed by
anti-arrhythmic drugs
Except in Rats, which require thousand times higher doses of
digitalis than man and other animals
33. Digoxin induced ventricular arrhythmia
Digoxin infused into jugular vein using
perfusion pump at the rate of 85 µg/kg
in 0.266 ml/min until cardiac arrest
Test drug given orally 1 hour or
intravenously 1 min prior to digoxin
infusion
The period until onset of ventricular extra systole, ventricular
fibrillation and cardiac arrest
Amount of infused digoxin required to induce ventricular
fibrillation
34. Digoxin induced ventricular arrhythmia
Sodium channel blocking drugs:
Diisopyramide, Lidocaine, Flecainide
Intravenous Amiodarone
Calcium channel blockers except Magnesium sulphate
Beta blockers
Hashimoto K. Arrhythmia models for drug research: classification of antiarrhythmic drugs. J Pharmacol Sci. 2007 Apr;103(4):333-46.
35. Strophanthin or Ouabain induced
ventricular arrhythmia
Male or female dogs weighing 20 kg used
Strophanthin K: continuous IV infusion at a rate 3 µg/kg/min
Test drug given when arrhythmia become stable for 10 min, either
Intravenously in dose between 1.0 to 5.0 mg/kg OR
Intraduodenally in dose between 10 to 30 mg/kg
Test drug considered to have an anti-arrhythmic effect if extra
systoles disappear, Immediately after i.v. administration OR
within 15 min after i.d. administration
Standard drugs: Quinidine ( 3 mg/kg i.v.) and
Lidocaine (10 mg/kg i.d.)
36. Other arrhythmogenic agents
Halothane – Adrenaline arrhythmia
Calcium chloride / Barium chloride
Benzene vapours
Grayanotoxin I, a biologically active diterpenoid
37. Electrically induced arrhythmias
Ventricular fibrillation electrical threshold
Programmed electrical stimulation induced re-entry
arrhythmia in old myocardial infarction in dogs
38. Ventricular fibrillation electrical threshold
Single pulse stimulation
Train of pulses stimulation
Continuous 50-hz stimulation
Sequential pulse stimulation.
39. Ag - AgCl stimulating
electrode embedded
in a Teflon disc
Anodal constant current
pulses for 400 ms
0.2- to 1.8-second train
of 50-Hz pulses is
delivered 100 ms after
every 18th basic
stimulus
40. Ventricular fibrillation electrical threshold
Current intensity increased in increments of 10 µA to 1.0 mA or
until ventricular fibrillation occurs.
VFT is the minimal current intensity of the pulse train required
to induce sustained ventricular fibrillation
The heart is allowed to recover to control conditions for 15 to 20
min.
Test drugs are administered through the femoral vein
VFT determined before and after administration of test drugs &
compared
41. Programmed electrical stimulation induced re-
entry arrhythmia in old myocardial infarction
Dogs (8 – 12 kg) anaesthetized
Thoracotomy
Heart exposed, LAD isolated
Ligature tied around artery & needle
Needle removed
Critical stenosis of vessel
Reperfusion for 2 hrs in presence of
stenosis
42. Programmed electrical stimulation induced re-
entry arrhythmia in old myocardial infarction
Epicardial bipolar electrode sutured on
intraventricular septum
Silver disc electrodes implanted s.c for ECG
Thoracic incision reopened after 6 – 9 days
Programmed electrical stimulation
performed3 premature extra stimuli applied to induce VT and VF
If sustained VT / VF occur, epicardial defibrillation performed to stop
them.
After 2 control runs, test drug given as IV bolus and arrhythmia
induced by same protocol.
The minimum current intensity of pulse required to sustain VF is
recorded before and after administration of test drugs
Also, drug effect can be demonstrated by change of severity of
induced arrhythmia before and after drug administration
Eg. K channel blocker Dofetilitde decreased severity of
arrhythmia,
VF (control) PVC / No arrhythmia (treated)
Used to demonstrate even the pro-arrhythmic potential of drugs
Eg: Flecainide has shown to aggravate the EPS induced
arrhythmia compared to control
43. Mechanically induced arrhythmia
Characteristics of collateral circulation of different animal species
Automaticity induced ectopic beats and subsequent re-entry
movement of excitation.
Michiel JJ, Tobias Opthof, Andre GK. Animal models of cardiac arrhythmias. Review. Cardiovascular Research 39 (1998) 165–177
44. Reperfusion arrhythmia in rats
Equilibration time of 45 min
Test drug/ vehicle/control IV
ligature closed for 15 to 90 min
Reperfusion for 30 min
ECG
Lead II
5 min
45. Reperfusion arrhythmia in rats
Ischaemia activates ATP dependent
K channel + Shorten AP
K channel blockers
Ischaemia stops Na/K ATPase
Increase intracellular Na
Na/Ca exchange
Intracelluar Ca overload
Na channel blockers
Ca channel blockers
46. Reperfusion arrhythmia in rats
1. Mortality
2. Hemodynamics (HR, BP )
3. Arrhythmias
Ventricular extrasystoles (PVC)
o Percent animals with PVC
o Number of PVC/5 or 30 min
Ventricular tachycardia
o Percent animals with VT
o Duration of VT/ 5 or 30 min
Ventricular fibrillation
o Percent animals with VF
o Duration of VF/ 5 or 30 min
4. Infarct size (using p-nitro
blue tetrazolium chloride
staining )
47. Reperfusion arrhythmia in Dogs
Harris (1950) : Mortality in dogs was lower with 2 stage
ligation procedure than 1 stage ligation
Canine two stage coronary ligation
arrhythmia model
(Partial f/b complete occlusion)
Consistent and stable sub acute to chronic ischaemic
arrhythmias, showing ventricular tachycardia but never
deteriorating to VF – Late Arrhythmias
48. Canine two stage coronary ligation
arrhythmia model
Two ligatures placed around the artery and a 21 guage needle
First ligature tied around artery and needle and removed
Second ligature tied around artery tightly
Chest closed and dog allowed to recover
Baseline observations when dog is conscious
(Lead II and avL of ECG, BP)
Test drug/ vehicle/ control administered
30 min
22 – 24 hour later Arrhythmias develop
30 min
49. Canine two stage coronary ligation
arrhythmia model
Arrhythmic ratio: Number of PVC over a predefined time
Number of total beats in that time
Advantages:
VT becomes fulminant 24 hours later in conscious state,
lasting up to 48 hours
Drugs given in conscious state, so CNS side effects can be
evaluated while measuring anti-arrhythmic efficacy
Best model to evaluate a potential Na channel blocking drug
Effective plasma conc of drug to decrease the ratio to
50% can be calculated.
50. Sudden coronary death model in dogs
LAD occluded for 2 hours resulting in critical stenosis
Reperfusion for 2 hours
Silver electrode passed through lumen of left circumflex
coronary and sutured
Test drug given after dogs recover (day 3 – day 5)
Direct anodal 15 µA current from a 9 V battery is applied to the
silver electrode
Lead II ECG recorded for 30 sec every 15min
24 hours later
Animal sacrificed
Heart and thrombus mass in LCX removed and weighed
1. Time to onset of ventricular ectopy and lethal arrhythmia
studied (Non sustained & sustained arrhythmias evaluated)
2. Heart sections stained with Tetrazolium triphenyl chloride
TTC to identify infarct areas.
Non-sustained VT: 5 or more repetitive ventricular responses
initiated but terminated spontaneously
Sustained VT: persist for at least 30 sec or require ventricular
burst pacing for its termination
53. In c/o prior MI or ongoing ischemia, Class I effect was
accepted as potentially pro-arrhythmic
Perceived necessity of demonstrating the safety of such
drugs in large placebo controlled trials in patients with
underlying cardiac pathology and left ventricular systolic
dysfunction
Cardiac Arrhythmia Suppression Trial (CAST)
54. PHASE I
Safety, tolerability and PK/PD parameters
Primary outcome.
Safety:
Spontaneous reporting of AE,
clinical examinations,
lab tests
PK:
Cmax, T max, AUC, t1/2
55. PHASE II STUDIES
Short term study:
Efficacy and safety of test drug in AF and Afl
Long term study:
Conversion and maintenance sinus rhythm in persistent AF
and AFI
Prophylactic treatment of paroxysmal atrial fibrillation or
atrial flutter
56. Short term study
Efficacy and Safety of IV bolus administration of test drug vs.
placebo and reference drug in the acute termination of atrial
fibrillation and flutter
Incidence of conversion to SR within pre defined time T
Incidence of side effects
AF/AFI
> 6
hours
but < 60
days
A
B
C
D
Y
P
P vs. A (lowest dose) vs. B (Second
lowest dose) vs. Y
P vs. C (Second highest) vs.
D (highest dose) vs. Y
I.V.
58. Long term study
Conversion & maintenance sinus rhythm in persistent AFI or AF
To achieve conversion of persistent AF or AFl to SR within 5 half
lives of the start of drug dosing
Incidence of conversion to SR within 5 half lives
To maintain sinus rhythm following electrical or chemical
cardioversion
Time to first recurrence of AF or Afl lasting atleast 24 hrs
Efficacy vs. Placebo & vs. Reference standard
Safety at the most effective dose compared to placebo
Incidence of serious side effects
59. Conversion & maintenance sinus rhythm in persistent AFI or AF
Long term study
AF/AFI > 2 weeks
but < 26 weeks
Hospitalized
Randomized
A
B
C
Y
P
12 lead ECG daily
QTc interval > 15 % over
baseline, dose is halved
QTc interval exceeds 550 msec
or > 25% over baseline, the
subject is withdrawn
Sinus
rhythm
No Sinus rhythm
Electrical
cardioversion
24 hours monitoring
Discharged & F/U till
Relapse of AF/AFI lasting 24 hours
OR Maintenance of SR for one year
61. Tools for assessment
Measure Description Score Comments /
Limitations
Symptom Checklist
– Frequency &
Severity Scale
Based on severity &
frequency of symptoms
0 – 64 for
frequency
0 – 48 for
severity
Most extensively
validated
Time consuming
University of
Toronto atrial
fibrillation severity
scale
14 item disease specific
scale
3 – 30 Validated &
reproducible
Time consuming
Canadian
cardiovascular
society AF severity
scale
Based on major
symptoms, symptom -
rhythm correlation,
impact on QoL
0 – 4 Simple & correlates
with SF – 36 QoL
scores
Poor correlation with
AF burden
Rienstra Michiel et al. Symptoms and Functional status of patients with atrial fibrillation: State of art and future research opportunities. Circulation. 2012 Jun;
125(23): 2933 – 2943
62. Tools for assessment
Measure Description Score Comments /
Limitations
Atrial fibrillation
scale
6 questions on dyspnoea
at rest, exertion,
limitations in daily life
0 – 60 Items based on
patient interviews
Satisfactory, reliable,
valid
European Heart
Rhythm
Association
classification
Based exclusively on
patient reported
symptoms
Class I – IV Simple
Not used in studies
yet.
No validation done
Rienstra Michiel et al. Symptoms and Functional status of patients with atrial fibrillation: State of art and future research opportunities. Circulation. 2012 Jun;
125(23): 2933 – 2943
63. Prophylactic treatment of paroxysmal atrial fibrillation or atrial flutter
Long term study
Efficacy of test drug at 2 doses vs. placebo vs. control in prolonging
the arrhythmia-free period
Time from first dose of study medication to first ECG-
documented symptomatic AF, AFl or SVT.
Study Design: Depends on incidence of pro-arrhythmia observed
in prior trials
Initiation of study drug using in-patient hospitalization,
continuous monitoring and a 12-lead ECG prior to each
morning dose OR
close out-patient monitoring and daily transmission of a rhythm
strip prior to morning dose.
64. Phase III: in patients at risk of sudden death
due to ventricular arrhythmias
Camm AJ, Pratt CM, Schwartz PJ, Al-Khalidi HR, Spyt MJ, Holroyde MJ, Karam R,Sonnenblick EH, Brum JM; AzimiLide post Infarct surVival Evaluation
(ALIVE)Investigators. Mortality in patients after a recent myocardial infarction: a randomized, placebo-controlled trial of azimilide using heart rate variability for
risk stratification. Circulation. 2004 Mar 2;109(8):990-6.
65. Ischemic/
non-ischemic
CM
(EF 36 -
50%)
Phase III: in patients at risk of sudden death
due to ventricular arrhythmias
To evaluate the effects of test drug at 2 doses versus placebo on
all cause mortality in subjects at low to moderate risk of SD
Death due
to any cause
Double-blind,
placebo-
controlled,
parallel design
Decreased / Unaffected
Mortality
Increased Mortality
Drug use in Normal hearts with SVTDrug use in mod. LV Dysfunction
66. Species differences do exist, no animal model will accurately
mimic the human patient suffering from, or threatened by an
arrhythmia
Electrophysiological studies should be encouraged in animals
with ‘naturally’ occurring cardiovascular disease
Clinical trials of potentially useful antiarrhythmic drugs are at
the present time both complex and costly
Conclusion
67. Situation may improve in the future if other classes of drug
action are discovered which do not cause proarrhythmia as do
the class I and III compounds
Currently, the attention is mainly towards ongoing development
of implantable defibrillators in the prevention of SD due to
ventricular arrhythmias, and in the case of atrial arrhythmias, to
the enormous strides being made in catheter-based ablation
techniques.
Conclusion
Editor's Notes
CARDIAC AP
AP generated from autoarrhythmic cells create waves of depolarization that spread through the contractile cells via gap junctions. After depolarization, the contractile cells slide over each other resulting in contraction.
Arrhythmia consist of cardiac depolarization that deviate from the normal in one or more aspects: there is a abnormality in site of origin …
Understanding the action potential of cardiac muscle is necessary for the understanding of basic pharmacology of anti-arrhythmic drugs.
Eg. Beta blockers , vagal discharge decrease the normal pacemaker rate by increasing the diastolic interval
Whereas, hypokalemia, Beta agonist, acidosis cause tacharrhythmias by decreasing diastolic interval and hence decresasing interval between the two depolarization.
A common abnormality of conduction is “rentry” (circus movement) in which impulse reenters and excites areas of heart more than once.
For re entry to occur, 3 conditions must co-exist.. .. (picture)
Slowing of conduction may be due to depression of sodium or calcium current or both. So, drugs that abolish re-entry usually work by further slowing depressed conduction (by blocking sodium / calcium channels) and causing bidirectional block.
Secondly, increasing the refractory period will also make re-entry unlikely. Longer the RP near the site of block, greater is the chance that tissue will be refractory when re-entry is attempted.
The main aim of therapy is to reduce the ectopic pacemaker activity and modify the conduction / refractoriness in re-entry circuits.
The major pharmacological mechanisms currently available to accomplish these goals are,
Thus, these drugs block electrical activity when there is a tachycardia ( many channel activation and inactivations per unit time) or when there is a significant loss of resting potential. And electrical activity in normally polarized part of the cell is minimally affected.
So, now, the most widely used classification of anti-arrhythmic drugs recognizes four classes..
Bretylium, Ibutilide, Doefetilide, Amiodarone, Sotalol
Present Indications for Antiarrhythmic Drug Therapy
Currently, the major indications for antiarrhythmic drug use are atrial fibrillation (AF) and atrial flutter. Antiarrhythmic drugs are used acutely as an alternative to electrical
cardioversion for both persistent AF and AFl so as to achieve sinus rhythm (SR) and chronically to prevent recurrence. They are also used to diminish the frequency of paroxysmal or spontaneously terminating episodes of AF and Afl. Antiarrhythmic drugs are now mainly used for these arrhythmias only
while awaiting definitive curative ablation
ventricular arrhythmias: Implantable defibrillators have been demonstrated to be superior to antiarrhythmic drugs .
Antiarrhythmic drugs have so far not been successful when used as primary prevention of ventricular arrhythmias in patients at risk of SD. At best, the drugs studied to date
with Class III effects have neutral effects on all-cause mortality in the post-MI patient population.
Clearly, there is a need for drugs which are more effective and safer in terms of pro-arrhythmic potential and organ toxicity than those currently available
Ventricular arrhythmia, especially torsades des pointes can be evaluated using isolated ventricular myocytes.
Guinea pig ( 250 – 350 g) are sacrificed. Heart removed and it is perfused with solution containing collagenase and protease. This digests the heart and then the heart is cut into small pieces. Then one piece is placed in HEPES buffered saline and shaken till individual cells are isolated. The trans-membrane potential is recorded using a glass electrode. The isolated heart fiber is stimulated electrically. First during control period the muscle fiber is stimulated at a frequency of 3Hz and the AP is recorded. Then the test drugs are added in the medium and the fiber is stimulated again and the AP recorded. The change in AP amplitude before and after is compared between test and control.
Perfused with,
- Oxygenated calcium free HEPES buffered saline at 37 C at the rate 10 ml/min for 5 min
Same solution containing type II collagenase and type XIV protease for 8 min
HEPES buffered saline containing 0.2 mM calcium chloride for 5 min
Action potential measured during control and after perfusion with two successively increasing concentration of a drug
The change in AP amplitude before and after is compared
The anti-arrhythmics are divided in to 4 different classes depending on their mode of action. These electrophysiological actions also have functional manifestations… like.. Sodium channel blockade decreases excitability, Potassium channel blockade lengthens the refractory period and calcium channel blockade decreases tension of cardiac muscle, which forms the basis of a simple and accurate in vitro method to identify and classify the potential anti arrhythmic drugs into class I, II, III and IV.
Guinea pigs (Either sex, weighing 200 – 400 gm) are stunned, carotid arteries are severed and thoracic cage opened immediately. The heart is removed and placed in a container of prewarmed, preoxygenated physiological solution. The heart is pinned to the dissection tray, right ventricle is opened, and the tendinous end of the papillary muscle is ligated with a silk thread and other end is clamped into a tissue holder, at the end of which is a platinum wire field electrodes.
the period for which no contraction develops is the ERP
In 1897, Oscar Lagendorff established the isolated perfused mammalian heart preparation, which was considered a breakthrough in cardiovascular research. The basic principle is that heart is perfused in a retrograde direction from aorta at constant pressure or constant flow with oxygenated saline solutions. Retrograde perfusion closes the aortic valves just as in the in-situ heart during diastole, so that the perfusate then enters the coronary arteries, flowing off the coronary sinus and the opened right atrium.
I will be describing the use of Lagendorff preparation in the evaluation of anti-arrhythmic drugs.
Guinea pigs of either sex weighing 200- 400 gram are anaesthesized with 50 mg/kg of sodium Thiopentone and mounted on the dissection tray.
The thoracic cage is opened and the beating heart is exposed. The pericardium is opened and aorta is located and cut below the point of division. The cannula is then inserted into the aorta and tied and the heart is perfused with the oxygenated perfusate (Ringer’s solution). The perfusate is allowed to flow freely at constant pressure by opening the stopcock fully.
The preparation is transferred to a double wall plexiglass apparatus at 37 C . This essentially completes the Lagendorff preparartion.
A ligature is placed around the LAD coronary artery and occlusion maintained for 10 min followed by reperfusion. An epicardial ECG electrode is used for pulsatile stimulation and induction of arrhythmias.
And, two electrodes A and B are placed on the apex and base of the heart to record an electrogram.
Test drug is administered through the perfusion medium either before or after occlusion.
The incidence and duration of ventricular tachycardia or ventricular fibrillation is recorded in the control as well as test group.
[ The experiment is started with stimulant drugs (adrenaline, NA, calcium) followed by depressant (Ach, potassium) and finally antagonists..as the effect of antagonists take a long time to wear off.
Apart from guinea pigs, even Newzealand rabbits and albino rats can be used for this preparation; Perfusate is cold kerb’s solution in rats and guinea pigs and McEwens solution in case of rabbit heart.
Rabbit heart can beat up to 9 hour if not treated with toxic drugs.]
Acetylcholine indirectly opens Potassium Channel in Cardiac Muscle and fibrillation is produced when atria are exposed to Ach or Potassium chloride
ECG recorded on a kymograph.
Control arrhythmia are produced and allowed to continue for 6-10 min. After a 30 minute rest period, arrhythmia is again induced and test drug is added to the bath after 3 minutes.
Test compound is found to be effective if arrhythmia disappears immediately or within 5 minutes after addition of test drug to the organ bath.
Recording an electrocardiogram is an essential tool in the evaluation of anti-arrhythmic drugs. Similar to heart rate, the ECG is different between various species.
The lead placement is shown here.
Lead II used most commonly, as it is in line with the anatomical axis of the heart, between right foreleg and left hindleg
Lead I is between right and left foreleg, is stated to lie in the axis of horizontal heart
Lead III is between left foreleg and left hindleg and in line with the vertical heart.
Also, unipolar leads designed as avl, avr and avf may be used.
In case of male SD rats, the animal is anesthetized by intraperitoneal injection of 60 mg/kg pentobarbitone. The right jugular vein is cannulated for injections
The variables measure include:
In rats, it is difficult to detect a T wave and so T wave calculations are made on the basis of repolarization wave that follows QRS complex. And the RSh magnitude is taken as a measure of the extent of S wave depression …as exerted by Class I anti-arrhythmics.
Earlier the in vivo models were classified based on the segment of heart involved.. Atrial and Ventricular arrhythmia models.
But, now, the arrhythmogenic stimuli can be divided into 3 groups…
A large number of agents alone or in combination are capable of inducing arrhythmias. Administration of anaesthetics like chloroform, halothane, ether, which act as sensitizing agents, followed by a precipitating stimulus such as IV adrenaline or cardiac glycosides, aconitine cause arrhythmias.
The sensitivity of these arrhythmogenic substances differs among various species.
The plant alkaloid, Aconitine, persistently activates the sodium channels resulting in ventricular arrhythmias. So, drugs considered to have anti-arrhythmic properties can be tested in Aconitine intoxicated rats.
anaesthetized with Urethane 1.25 g/kg IP)
The Anti-arrhythmic effect of a test compound is measured by the amount of Aconitine/100 g of animal which induces,
Ventricular extrasystoles, Ventricular tachycardia, Ventricular fibrillation and Death
Procainamide and Lignocaine at their screening dose of 5 mg/kg increased LD100 by 65%.
LD 100 is the lethal dose.. Dose having 100% probability of death.
Thus, this model has been proven to be valuable in screening of drugs for anti-arrhythmic activity.
The cardiotonic steroid digitalis is arrhythmogenic and its effect can be explained by intracellular calcium overload… due to…
Na –K – ATPase inhibition causing intracellular Na accumulation, followed by,
Intracellular calcium accumulation by enhanced Na/Ca exchange…. Leading to arrhythmias..
The exact mechanism by which Ca overload cause delayed after depolarizations or abnormal automaticity is still not known.
Male guinea pigs ( Marioth strain) weighing 350 – 500 gram is anaesthetized with pentobarbital sodium.
Trachea, jugular vein, carotid artery is catheterized and the animal is maintained on artificial respiration. Blood pressure recorded through the carotid artery and ECG recorded through subcutaneous needle electrodes.
The variables recorded are…
The variables after treatment with anti-arrhythmic drugs are compared statistically with controls receiving digoxin only.
The sodium channel blocking drugs suppressed this digitalis induced arrhythmias.. The effective plasma concentration were found to be closely related to the sodium channel blocking concentrations. Also, IV Amiodarone was effective probably due to sodium channel blocking property.
But, Calcium channel blocking drugs did not suppress this arrhythmia even though the mechanism of generation of arrhythmia is though to be intracellular calcium overload. An exception was Magnesium sulphate, which acts as a Ca channel blocker in vivo.
As regards the ineffectiveness of Ca channel blockers, its hypotensive effect have made it impossible to increase the doses high enough to decrease the influx of Ca to myocardium. So, when intracoronary administration of high dose verapamil was tried, PVC was effectively suppressed.
Similarly, beta blockers were not effective unless high dose to suppress sodium channels were given.
Similar to previous model, Strophanthin K is a cardiac glycoside that induces ventricular tachycardia and multifocal arrhythmia in dogs.
This model is very much similar to digitalis induced ventricular arrhythmia, with few important points to mention..
Male or female dogs weighing 20 kg used. The test drug can be given by intravenous or intraduodenal route by cannulating the duodenum.
The test drug is administered in increasing doses at 15 min interval, and is considered ineffective if it does not improve arrhythmia within 60 min following drug administration.
Some other substances which can be used as arrhythmogenic agents are..
Normal pacemaker activity of heart is under the control of sympathetic and vagal influences and sympathomimetic drugs are known to increase the pacemaker activity. Some anaesthetics like halothane are known to sensitize myocardium to catecholamines. Thus, under the influence of halothane, adrenaline in low doses induce severe VT.
The use of anti-arrhythmic drugs in the treatment of ventricular arrhythmias aims to prevent the development of ventricular fibrillation.
Several electrical stimulation techniques have been used to measure ventricular fibrillation threshold such as single pulse stimulation, train of pulses stimulation, continuous 50-Hz stimulation and sequential pulse stimulation.
Adult dogs weighing 8–12 kg are anesthetized with sodium pentobarbital (35 mg/kg) and maintained on artificial respiration with monitoring of BP and temperature. The chest is opened by a midline sternotomy and the heart suspended in a pericardial cradle. The sinus node is crushed and a 2.0 mm diameter Ag-AgCl stimulating electrode is embedded in a Teflon disc sutured to the anterior surface of the left ventricle. is applied through the electrode for 400 ms. Electrical stimulation is programmed through a digital stimulator.
A recording electrode is placed on surface of each ventricle and Lead II of ECG is monitored.
To determine ventricular fibrillation threshold (VFT), a 0.2- to 1.8-second train of 50-Hz pulses is delivered 100 ms after every eighteenth basic driving stimulus
The current intensity increased from the diastolic threshold in increments of 10 µA to 1.0 mA or until ventricular fibrillation occurs.
VFT is the minimal current intensity of the pulse train required to induce sustained ventricular fibrillation
The heart is allowed to recover to control conditions for 15 to 20 min.
Test drugs are administered through the femoral vein
VFT determined before and after administration of test drugs & compared
This arrhythmia is produced by applying premature extra stimuli in dogs who are operated by two stage coronary ligation 6- 10 days prior and the mechanism of generation is thought to be re-entry around the scar tissue of the old infarction.
Dogs weighing 8 – 12 kg are anaesthetized with pentobarbital sodium, maintained on artificial respiration. Thoracotomy performed and heart is exposed
Programmed electrical stimulation performed through the electrode implanted on non infarcted zone.
The rank of arrhythmia was used to evaluate drug effects and the order by their severity is, VF is the most severe f/b sustained VT, non sustained VT, PVC and no arrhythmia.
Dofetilide works by selectively blocking the rapid component of the delayed rectifier outward potassium current . This causes the refractory period of atrial tissue to increase, hence its effectiveness in the treatment of atrial fibrillation and atrial flutter.
Due to this property, this model was used to demonstrate even the “ proarrhythmic potential of drugs like Na channel blockers”
But.. It is difficult to judge whether this arrhythmia model is appropriate and selective one to differentiate or select dangerous Na channel blockers
Arrhythmias can be induced directly by ischemia or by reperfusion. The generation mechanism of arrhythmia is thought to be automaticity induced ectopic beats and subsequent re-entry movement of excitation.
For this model, various animals could be used EXCEPT Guinea pigs. Because the collateral circulation is highly developed in guinea pigs and hence ligation of one of the main branches would make it impossible to reproduce situations of myocardial ischemia.
I will explain the reperfusion model in SD rats.. Because in “pigs” not guinea pigs…. and rats, spontaneous arrhythmia occurs in nearly 100% cases. Also, in small animals like rats, the mortality is less, the VF often reverts to sinus rhythm. A similar model can be done in dogs as well.
The rationale behind this model is that left main coronary artery ligation results in ventricular arrhythmia and myocardial infarction. ECG is recorded during ligation and reperfusion.
The amount of infarcted tissue is measured by means of p-nitro blue tetrazolium chloride staining in myocardial sections.
And this model mimics myocardial ischemia related arrhythmias that are frequently observed.
SD rats weighing 350 – 400 g are anaesthetized with pentobarbitone sodium 60 mg/kg IV. ( Why rats? – because in “pigs” not guinea pigs..and rats, spontaneous arrhythmia occurs in nearly 100% cases. Also, in small animals like rats, the mortality is less, the VF often reverts to sinus rhythm). The animal is maintained on artificial respiration. BP recorded from carotid artery using a pressure transducer. Jugular vein is cannulated for administration of test drug. The chest opened and heart exposed.
The chest is opened and heart exposed. A thin silk thread attached to a atraumatic needle placed around LAD 2-3 mm distal to its origin for later ligation. After an equilibration time of approx. 45 min, the test drug/ vehicle/control is administered IV. 5 min later, ligature closed for 15 to 90 min and subsequently reperfused for 30 min.
If any animal develops dysarrhythmia or sustained fall in BP to less than 70 mm Hg by the procedure itself has to be discarded from the study.
Ischemia activates ATP dependent K channel and shorten duration of A, so K channel blockers with a opposite effect to prolong AP should be effective.
Also, Iscahemia stops action of Na/K ATPase followed by an increase in the intracellular Na concentration, which results in intracellular Ca overload. Thus, these exchange blockers are expected to overcome this arrhythmia.
Some Na channel and K channel blockers have been reported to be effective in suppressing coronary occlusion/reperfusion arrhythmias.
The variables evaluated are…
Changes in parameters in drug treated animals are compared to vehicle control values.
A similar model can be done in dogs as well.
A similar model can be done in dogs as well.
Here’s an example of a experiment using this model, wherein , 10 out of 12 control dogs died from ventricular fibrillation during the 30 min reperfusion period.
Harris (1950) : Mortality in dogs was lower with 2 stage ligation procedure than 1 stage ligation. Therafter, the Canine two stage coronary ligation arrhythmia model
Was reported.
Which is characterized by consistent and stable sub acute to chronic ischaemic arrhythmias, showing ventricular tachycardia but never deteriorating to VF resembling late arrhythmias.
Now why in dogs?
Because, it has been shown that the success rate of obtaining arrhythmia for drug evaluation in high, nearly 90% in dogs.
The number of sinus and ectopic beats are calculated for each successive 5 min period.
The arrhythmic ratio is calculated. (For example, the arrhythmic ratio is 0 during the sinus rhythm, while it is 1 during ventricular tachycardia). Effective plasma conc of drug to decrease the ratio to 50% can be calculated.
The main advantages of this model is that, very stable arrhythmias develop after several hours and VT becomes fulminant 24 hr later in conscious state, lasting up to 48 hours. SO, this arrhythmia can be used to evaluate and compare drug effects.
Sudden coronary death is one of the leading causes of death in developed countries.
The important features in this model are… In the procedure.. The animal is dissected while on artificial respiration as described before. But, here, ..
Tetrazolium triphenyl chloride
Ventricular tachyarrhythmias are defined as ‘non- sustained’, if five or more repetitive ventricular responses are initiated reproducibly, but terminated
spontaneously. Ventricular tachyarrhythmias are defined as ‘sustained’, if they persist for at least 30 s or, in the event of hemodynamic compromise, require
ventricular burst pacing for their termination.
The development of new antiarrhythmic drugs has become a major challenge since the publication of the Cardiac Arrhythmia Suppression Trial (CAST) results in 1989.
This was the first study designed to demonstrate that antiarrhythmic drugs with a potent Class I effect (flecainide and encainide) decrease mortality as compared to placebo in patients with remote myocardial infarction (MI) and frequent ventricular ectopic beats, who are therefore at increased risk for sudden death (SD) due to malignant ventricular
arrhythmia.
Improved survival was expected as these drugs markedly suppressed premature ventricular contractions in the same study patients.
But, Instead, an increase in mortality was observed on antiarrhythmic drug compared to placebo.
This result affected antiarrhythmic drug development in two ways.
In the setting of prior MI or ongoing ischemia, Class I effect was now generally accepted as potentially proarrhythmic. Instead, attention was directed to drugs which prolong action potential duration and refractoriness.
Phase 1 :
Aim. Safety, tolerability and PK/PD parameters.
Healthy volunteers.
Primary outcome.
Safety: Spontaneous reporting of AE, clinical examinations, lab tests
PK: Cmax, T max, AUC, t1/2
All usual
This study is often the first type of study done as it involves only single acute intravenous administration of drug and therefore requires only short-term monitoring of the subject.
subjects with AF or AFl of duration longer than 6 hours but less than 60 days will be randomized. Episodes of AF/AFl of shorter duration are likely to convert to SR on placebo while episodes of very long duration are less likely to respond to drug therapy.
Subjects who meet the study inclusion/exclusion criteria will be randomized in double-blind fashion to investigational drug X at dose A, B, C or D, the reference drug Y or placebo.
In order to assure safety of the study drug at the doses proposed, such a study could be performed in a tiered fashion. If safety is shown to be an issue at the higher doses, the study would continue with only those doses judged to be safe.
I would like to emphasize here that he usual initial clinical approach to a patient presenting with AF/AFl should be followed, i.e., anticoagulation as recommended by current clinical guidelinesand control of the ventricular response rate (6) before initiating study drug
An example of such a study have been performed in the development of such Class III drugs as dofetilide and ibutilide .
Comments:
If safety and efficacy are demonstrated in such a study, other Phase II studies can be considered. If safety is demonstrated but there is little efficacy of I.V. drug, still there is a possibility that the test drug may be very effective in preventing recurrence of arrhythmia.
But, if safety is clearly an issue, there will be little impetus to pursuing further drug testing.
long-term Phase II studies are initiated in a monitored setting in hospital for at least the duration of time necessary to achieve steady-state.
Also, evaluation of the efficacy of test drug in the context of a long-term oral dosing protocol should be done separately in patients with cardiac pathology and EF < 35 % as compared to those with better preserved EF or with normal hearts.
I will brief give u an outline of long term study in patients with normal hearts or fairly well-preserved EF (> 35%) despite cardiac pathology.
WHY ?? I will tell you later..
Primary objectives:
a. To determine the efficacy of oral administration of test drug at the highest tolerated dose compared to placebo and to reference drug Y in achieving conversion of persistent AF or AFl to SR within 5 half lives of the start of drug dosing (or within the time to reach maintenance plasma levels if loading doses are used).
b. To determine the efficacy of chronic oral administration of test drug at the highest tolerated dose compared to placebo and to reference drug Y in maintaining SR following electrical or chemical cardioversion.
To determine the safety of oral administration of the test drug at the most effective dose compared to placebo in subjects with persistent AF or AFl.
This is achieved through the study end points of….
STUDY DESIGN : FLOWCHART
Patients with AF or AFl of duration longer than 2 weeks but less than 26 weeks will be recruited. Subjects who meet the study inclusion/exclusion criteria are admitted in hospital for the duration of time necessary to reach the plateau effect of test drug or reference drug Y whichever is longer.
Randomization is done in a double-blind fashion to oral investigational drug X at doses A, B, or C, the reference drug Y or placebo
A 12-lead ECG is obtained just before the first dose on all days while in hospital. If the QTcinterval is found to increase by > 15 % over baseline, the dose is halved. If QTcinterval exceeds 550 msec or increases by > 25 % over baseline, the subject is withdrawn from the study.
In subjects who are exposed to drug for a duration of time to achieve plateau levels and have not converted spontaneously to SR, electrical cardioversion is attempted.
In subjects who are exposed to drug for a duration of time to achieve plateau levels and have not converted spontaneously to SR, electrical cardioversion is attempted. Following conversion to SR, subjects are monitored for an additional 24 hours.
Subjects who acheieve SR are discharged from hospital on the same dose of study drug as last given in hospital and followed up regularly till one of the studyend points is reached: relapse to AF or AFl for at least 24 hours, documented by ECG, or maintenance of SR for one year.
Demonstration of efficacy and safety in such a study suggests the possibility of beginning drug on an outpatient basis.
[A 12-lead ECG is obtained at each visit, reviewed by the investigator and sent to a central facility which
generates a report including an interpretation of the patient’s rhythm and interval calculations. If the
investigator and central ECG facility interpretations do not agree with respect to possible AF, AFl or SVT,
the report should be sent to an Event Committee for a final decision. ]
Such an approach has been taken with both dofetilide in a trial called..SAFIRE D study
The various scoring systems to assess the secondary outcome measures are..
With the accumulation of data from studies on the acute conversion of AF and AFl with I.V. or oral investigational drug and the prevention of recurrence of arrhythmia, it is usually possible to identify a dose range which is effective and tolerated, so that drug groups can now be limited to one or two doses for evaluating efficacy and
safety when used for other indications, such as the prevention of paroxysmal AF (PAF).
This is randomized, double-blind, placebo-controlled, parallel-group clinical study.
This primary objective can be assessed separately in two strata:
• subjects with ischemic heart disease (IHD) and/or CHF (EF >35%)
• subjects with neither of the above
I will explain this with the help of a example..
A phase III mortality trial ALIVE compared azimilide to placebo in patients with recent MI (6-42 days), who were at moderate to high risk for sudden death as predicted by poor LV systolic function (EF < 35%). No difference in overall mortality was observed.
It is unlikely, however, that such placebo-controlled mortality studies in moderate to high risk populations will continue to be performed. The recent publication of MADIT II (Second Multicenter Automatic Defibrillator Implantation Trial ) and SCDHeFT results have shown the superiority of implantable defibrillators versus placebo in patients with poor LV function and IHD as well as versus placebo and amiodarone in non-ischemic CM. So, long-term mortality trials of test drug versus placebo are no longer ethical in this population.
Instead, the focus will likely be shifted to that group of patients with only moderately or minimally depressed LV function (EF 36-50%). These patients, while less at risk for SD from ventricular arrhythmias as a result of the underlying cardiac pathology, may however be at risk for greater mortality on investigational drug X if there is any
proarrhythmic potential of the new drug.
A typical plan of such a study would be.. With the objective of…
To evaluate the effects of investigational drug X at dose A versus placebo or dose B versus placebo on all cause mortality based on intention-to-treat in subjects at low to moderate risk of SD.
Subjects with ischemic or non-ischemic CM and LV EF of 36 - 50% will be enrolled.
if decreased or an unaffected mortality rate is demonstrated, this would permit drug use for atrial arrhythmias in patients with moderate LV dysfunction (EF 36-50%)
The demonstration of increased mortality in the presence of investigational drug in patients with underlying cardiac pathology and mild to moderate LV dysfunction would essentially limit the use of the new drug to patients with normal hearts experiencing supraventricular arrhythmias. Also, Even in this setting, long-term use should be frequently reevaluated as the patient ages and develops cardiac disease
